A sensitive method for light microscopic localization of brain receptors by in vitro autoradiography was developed previously in this laboratory. By this method we have mapped the locations of opiate receptors in the brains of rats and other vertebrates, including primates. Comparisons of radiolabeled opiate alkaloid binding with radiolabeled enkephalin binding have confirmed the existence of opiate receptor subtypes. A similar strategy led to the optimization of binding conditions to selectively reveal the distribution of substance P receptors and eledoisin binding sites. Immunohistochemistry is used to compare the distributions of putative neurotransmitters and their receptors. The relationship of these localization patterns with other markers of brain heterogeneity, provided by tract tracing and enzyme staining, allows hypotheses about functional circuitry in the central nervous system.